Optical device for the combination of light beams

ABSTRACT

An optical device for combining a first light beam and at least one second light beam includes a first beam splitting device, a second beam splitting device and a position detector. The first beam splitting device splits a first reference beam from the first light beam and a second reference beam from the second light beam. The second beam splitting device splits a third reference beam from the first light beam and a fourth reference beam from the second light beam. The position detector detects respective positions of the reference beams so as to enable a respective propagation direction and/or a respective position of the first and/or second light beams to be adjusted as a function of the detected positions of the reference beams.

The present invention relates to an optical device for combining a lightbeam and at least one further light beam.

In optics, a frequently occurring problem is to collinearly combinelight beams, in particular light beams of different wavelengths.

For example, in scanning microscopy, samples are often prepared with aplurality of markers, for example, a plurality of different fluorescentdyes, to simultaneously excite them with an illuminating light beamcontaining light of several excitation wavelengths. To produce theilluminating light beam, usually, the light beams of several lasers arecombined using usually a plurality of dichroic beam splitters arrangedin series. A scanning microscope having a dichroic beam combiner forinfrared and ultraviolet light is known, for example, from GermanPublished Patent Application DE 198 29 953 A1.

German Published Patent Application DE 198 35 068 A1 discloses amicroscope, in particular a laser scanning microscope, with illuminationover one wavelength and/or a plurality of wavelengths, where theintensity of at least one wavelength is controlled by at least onerotatable interference filter placed in the illuminating beam path, andwhere the at least one wavelength is at least partially reflected out ofthe illuminating beam path, and a plurality of filters for differentwavelengths can be arranged in series in the illuminating beam path.

In scanning microscopy, the light beams of a light source are coupledinto the scanning microscope and aligned to the optical path of thescanning microscope, and a sample is illuminated by the light beam toobserve the reflected or fluorescent light emitted by the sample. Thefocus of an illuminating light beam is moved in a sample plane using acontrollable beam deflection device, generally by tilting two mirrors;the deflection axes usually being perpendicular to one another so thatone mirror deflects in the X direction and the other in the Y direction.Tilting of the mirrors is brought about, for example, by galvanometerpositioning elements. The power of the light coming from the sample ismeasured as a function of the position of the scanning beam. Thepositioning elements are usually equipped with sensors to determine thecurrent mirror position.

In confocal scanning microscopy specifically, a sample is scanned inthree dimensions with the focus of a light beam.

A confocal scanning microscope generally includes a light source, afocusing optical system with which the light of the source is focusedonto a pinhole (called the “excitation pinhole”), a beam splitter, abeam deflection device for beam control, a microscope optical system, adetection pinhole, and the detectors for detecting the detection orfluorescent light. The illuminating light is coupled in via a beamsplitter. The fluorescent or reflected light coming from the sampletravels back through the beam deflection device to the beam splitter,passes through it, and is then focused onto the detection pinhole behindwhich the detectors are located. Detection light that does not derivedirectly from the focus region takes a different light path and does notpass through the detection pinhole, so that point information isobtained which leads to a three-dimensional image by sequential scanningof the sample. A three-dimensional image is usually achieved byacquiring image data in layers; the track of the scanning light beam onor in the sample ideally describing a meander (scanning one line in theX direction at a constant Y position, then stopping the X scan andstewing by Y displacement to the next line to be scanned, then scanningthat line in the negative X direction at a constant Y position, etc.).To allow the acquisition of image data in layers, the sample stage orthe objective is shifted after a layer has been scanned, and the nextlayer to be scanned is thus brought into the focal plane of theobjective.

The input coupling of the light beams for illuminating a sample into amicroscope is very critical with respect to alignment, especiallybecause the position and propagation direction of usually a plurality oflight beams of different wavelengths must exactly follow the nominaloptical path of the microscope. Alignment of a direct input coupling isfirst of all difficult, and secondly is usually not very reliablebecause, due to relatively long light paths, even the smallestvariations in the setup lead to imperfections requiring painstakingrealignment. Frequently, optical fibers are used to transport the lightbeams from the light source or light sources to the microscope in orderto reduce the problem to an alignment of the output coupling of theoptical fiber, which is in fact also painstaking, but, due to theshorter light paths, is less sensitive to misalignments. This does notsolve, but at best reduce the alignment problem, and involves otherdifficulties, such as the variation in the polarization direction of thelight beams.

The known systems for combining light beams of different wavelengthshave the disadvantage of being inflexible with respect to a change inwavelength. Moreover, it is not possible to ascertain whether thecombined beams actually propagate exactly collinearly. This effort isgenerally left to the user or to the service technician. If the lightbeams combined into an illuminating light beam are not substantiallycollinear, then abberations, in particuar artifacts and variations inbrightness, will occur in scanning microscopy.

It is the object of the present invention to propose an optical devicefor combining a light beam and at least one further light beam which canbe used flexibly, in particular for different wavelengths, and which atthe same time allows efficient and effective monitoring of the combiningof light beams.

This objective is achieved by an optical device which features a meansfor splitting a first reference beam from the light beam and a furtherfirst reference beam from the further light beam, as well as a furthermeans for splitting a second reference beam from the light beam and afurther second reference beam from the further light beam; the referencebeams being detectable by a position detector, and the propagationdirection and/or the position of the light beam and/or of the furtherlight beam being adjustable as a function of the detected positions.

Especially in scanning microscopy, the present invention has theadvantage of allowing the light beams of a light source or a pluralityof light sources to be aligned to the nominal optical path in a simpleand reliable manner. Also provided is an effective way of monitoring thealignment. In scanning microscopy, therefore, particularly stable imagequality is achieved by the avoidance of misalignments, while flexibleuse is possible in terms of the illuminating light wavelength.

In a preferred embodiment, the means for splitting off a first referencebeam is a first interface, and the further means for splitting off thesecond reference beam is a second interface. In one preferredembodiment, provision is made for a prism in which two of the lateralfaces form the first and second interfaces.

In another variant, an acousto-optical element is provided which can,for example, take the form of an acousto-optical modulator (AOM), anacousto-optical tunable filter (AOTF), or an acousto-optical deflector(AOD). In one preferred embodiment, the acousto-optical element canbring about the combination of the light beams. In another embodiment,the acousto-optical element is used for spectral separation and, forexample, is arranged upstream of a prism having a first and a secondinterface. The acousto-optical element can also be used to separatelyvary the optical power of the combined light beams and adapt it to theparticular use.

In a preferred embodiment, the position detector is calibrated fordifferent detectable positions. Each possible beam position ispreferably assigned a detectable set of positions; a corresponding setof nominal positions also existing for the nominal beam position. Thepropagation direction and/or the position of the light beams to bealigned are optimized until the nominal positions are detected. If thedetected positions deviate from the nominal positions, for example,because of an external disturbance, a realignment can be carried out bya control loop, so that the light beams to be aligned can be activelykept on the nominal optical path. Preferably, the first reference beamand the second reference beam are split off at different locations.

In one preferred embodiment, the means for splitting off a firstreference beam and the further means for splitting off the secondreference beam are parts of a single optical component, for example, twolateral faces of a prism. This embodiment is particularly stable andresistant to vibrations and shocks. The equipment or device according tothe present invention preferably has a compact monolithic design.Preferably, the splitting means and the position detector are arrangedin a fixed spatial relationship to each other, which providesexceptional stability because only a relative measurement needs to beperformed.

In one particular embodiment, the propagation direction and/or theposition of the light beam or beams can be changed by control elements,which can, for example, take the form of gimbal-mounted tilting mirrors.In a particularly preferred variant, it is proposed that the angles ofincidence and/or the locations at which the light beams strike the firstinterface be adjustable. Control elements are provided for this purposeas well. Possible control elements include all adjustable and preferablycontrollable light beam deflecting elements, for example, alsoacousto-optical deflectors (AOD). The control elements are preferablyplaced upstream of the means for splitting off a first reference beam.

Particularly advantageous is an embodiment in which the control elementscan be driven in open and/or closed loop as a function of the positionsdetected by the position detector(s). Such a design allowsimplementation of a closed-loop or open-loop control which automaticallyoptimizes the alignment of the light beam (light beams) and thecollinearity of the combined light beams.

In a preferred embodiment, the position detector is designed as a CCDdetector. It may also be designed as a photodiode line array, as aphotomultiplier array, or may also include a plurality of singledetectors. Preferably, the reference beams are detected together by oneposition detector, so that only relative measurements within theposition detector are required.

The position of the first reference beam and the position of the secondreference beam are preferably detected independently of each other andmade available for correcting the beam angles and positions. In apreferred embodiment, the positions of the reference beams are detectedsimultaneously, which is particularly advantageous in terms of externaldisturbances, such as vibrations and shocks, because the measurement isnot corrupted by spatial changes.

In one particular embodiment, the optical powers of the reference beamscan be determined independently of each other and used for correctingthe optical powers, which is important especially in scanningmicroscopy.

In a preferred embodiment of the alignment device, at least one furtherlight beam can be aligned to the nominal optical path. The light beamand further light beams can have different wavelengths.

In one preferred embodiment, a component for spectral separation isprovided upstream of the means for splitting off a first reference beam,preferably between the control elements and the means for splitting offa first reference beam. The component for spectral separation can, forexample, take the form of a plane-parallel plate, prism or grating.

In a different embodiment, a dispersive element is provided between themeans for splitting off a first reference beam and the further means forsplitting off the second reference beam.

In a preferred embodiment of the alignment device, the splitting meanssplits a further first reference beam from the further light beam, thefurther splitting means splits a further second reference beam from thefurther light beam; the further first and the further second referencebeam being detectable by the position detector, and the propagationdirection and/or the position of the second light beam being alignableto the nominal optical path as a function of the detected positions ofthe further first and the further second reference beam. Preferably, thepropagation direction and/or the position of the first and the furtherlight beam can be adjusted independently of each other. In one variant,further control elements are provided for adjusting the propagationdirection and/or the position of the further light beam. Preferably, thefurther control elements can be driven as a function of the detectedpositions of the further first and the further second reference beam.

The alignment device is particularly suitable for coupling light beamsinto a microscope, in particular a scanning microscope, which can bedesigned as a confocal scanning microscope. Accordingly, the nominaloptical path can be the optical path of a microscope, scanningmicroscope, or confocal scanning microscope. In another advantageousembodiment, the device serves at the same time for coupling out thedetection light emanating from the sample.

In one preferred embodiment, the light beams emitted by the light sourceinitially propagate collinearly and are separated both spatially andspectrally by a component for spectral separation upstream of the firstinterface. This embodiment is of interest, especially if the lightsource includes an optical fiber that transports all primary light beamstogether.

The subject matter of the present invention is schematically representedin the drawing and is described below with reference to the Figures, inwhich equally acting components are denoted by the same referencenumerals. Specifically,

FIG. 1 shows an optical device for combining at least two light beams;

FIG. 2 depicts an alignment device;

FIG. 3 shows a scanning microscope according to the present invention;

FIG. 4 illustrates a further alignment device.

FIG. 1 schematically shows an optical device 1 for combining at leasttwo light beams, namely a first light beam 3 and a second light beam 5.Light beams 3, 5 are emitted by a light source 7 including a first laser9 and a second laser 11. Light beams 3, 5 have different wavelengths.First light beam 3 strikes a first control element 13 including a firsttitling mirror 15 which can be tilted in two axes. Subsequently, firstlight beam 3 strikes a second control element 17 including a secondtitling mirror 19 which can be tilted in two axes. Second controlelement 17 directs first light beam 3 to a means for splitting off afirst reference beam 25; the means being designed as a first interface21 of a prism 23. At first interface 21, a first reference beam 25 issplit off by partial reflection and strikes position detector 27, whichis designed as a CCD array 29. After passing through first interface 21,first light beam 3 passes through prism 23 and strikes a further meansfor splitting off a second reference beam 33; the further means beingdesigned as a second interface 31. At second interface 31, a secondreference beam 33 is split off by partial reflection and, after totalinternal reflection at a third interface 35 and passage through firstinterface 21, it strikes position detector 27. Analogously, the path ofsecond light beam 5 is controlled by a third control element 37including a third tilting mirror 39 and a fourth control elment 41including a fourth tilting mirror 43. A further first reference beam 45is split off from second light beam 5 at first interface 21, and afurther second reference beam 47 is split off at second interface 31,the further first and second reference beams being directed to positiondetector 27. Located in front of the position detector is a lens 49,which focuses the reference beams onto CCD array 29. It is also possibleto provide for a slight defocus in order to achieve a better resolutionby interpolation across several pixels. From the various points ofincidence of the reference beams on CCD array 29 it is possible to inferthe locations and angles at which light beams 3, 5 strike firstinterface 21 and second interface 31, and thus the position andpropagation direction of light beams 3, 5 after exiting prism 23. Theposition detector generates position signals and transmits them to aprocessing unit 51. Based on the position data received, processing unit51 drives control elements 13, 17, 37, 41 until the light beams 3, 5exiting the prism are sufficiently collinear. The path differencebetween first reference beam 25 and second reference beam 33, andbetween further first reference beam 45 and further second referencebeam 47, respectively, is preferably about 20 mm, which results in achange in distance of about 20 μm on the position detector per mrad ofangular difference. 20 μm corresponds approximately to the distancebetween two pixels on conventional CCD detectors.

In the subsequent path of combined light beams 3, 5, provision is madefor an acousto-optical element in the form of an AOTF 53 to allow theoptical power of light beams 3, 5 to be adjusted separately. First lightbeam 3 and the reference beams 25, 33 split off therefrom are shown asdashed lines in the drawing. Second light beam 5 and the reference beams45, 47 split off therefrom are shown as dotted lines in the drawing.

FIG. 2 shows a device according to the present invention which isparticularly suitable for aligning a plurality of coaxial light beams 3,5 having different wavelengths to a common nominal optical path, forexample, after they are coupled out of an optical fiber, and to keepthem on the common path through suitable control.

Light beams 3, 5 strike a first control element 13 including a firsttitling mirror 15 which can be tilted in two axes and then a secondcontrol element 17 including a second titling mirror 19 which can betilted in two axes. Using control elements 13, 17, it is possible toadjust the position and propagation direction of light beams 3, 5. Afterpassing control elements 13, 17, light beams 3, 5 strike a firstplane-parallel plate 55, which separates light beams 3, 5 bothspectrally and spatially. Light beams 3, 5 are recombined by a secondplane-parallel plate 57. As a means for splitting off a first referencebeam 25 and a further first reference beam 45, second plane-parallelplate 57 has a first interface 21 which splits a first reference beam 25from first light beam 3, and a further first reference beam 45 fromsecond light beam 5. After passing through second plane-parallel plate57, light beams 3, 5 strike a means for splitting off a second referencebeam 33 and a further second reference beam 47, namely a secondinterface 31 which splits a second reference beam 33 from first lightbeam 3, and a further second reference beam 47 from second light beam 5.All reference beams are directed onto a position detector 27, which isdesigned as a CCD array 29.

The position detector 27 generates position signals and transmits themto a processing unit 51. Based on the position data received, processingwork 51 drives control elements 13, 17 until the light beams 3, 5exiting the second plane-parallel plate are in the desired position andpropagate in the desired direction. The current position and propagationdirection of light beams 3, 5 are permanently or regularly compared tothe desired position and propagation direction and, if necessary,automatically corrected by processing unit 51 via control elements 13,17.

FIG. 3 is a schematic view of a scanning microscope which is designed asa confocal scanning microscope. Light beams 3, 5 coming from a lightsource 7 in the form of a multiline laser are coupled by an opticalsystem 59 into an optical fiber 61 for transport. Output coupling is viaa further optical system 63 which substantially collimates light beams3, 5. The device below, whose mode of operation has already beendescribed with reference to FIG. 2, automatically aligns light beams 3,5 to the optical path of the scanning microscope.

After passing through illuminating pinhole 65, light beams 3, 5 are by abeam splitter 67 to a gimbal-mounted scanning mirror 69 which guideslight beams 3, 5 through scanning optical system 71, tube optical system73 and objective 75, and over or through sample 77. Sample 77 is labeledwith several fluorescent dyes. The detection light beam 79 emanatingfrom sample 77 passes through objective 75, tube optical system 73, andscanning optical system 71, and reaches beam splitter 67 via scanningmirror 69, and, after passing through detection pinhole 81, it strikes adetector 83 which designed as a multiband detector and generateselectrical detection signals which are proportional to the power ofdetection light beam 79. These signals are transmitted to PC 85. Thedetection signals are processed in PC 85 and displayed to the user on amonitor 87 as an image of sample 77. The scanning microscope isinsensitive to misalignments and allows quick and easy replacement ofthe light source or the optical fiber.

FIG. 4 shows a device for aligning a light beam 3 to a nominal opticalpath, which is illustrated in the drawing as a nominal optical axis 89.Light beam 3 strikes a first control element 13 including a firsttitling mirror 15 which can be tilted in two axes. Subsequently, firstlight beam 3 strikes a second control element 17 including a secondtitling mirror 19 which can be tilted in two axes. Second controlelement 17 directs first light beam 3 to a means for splitting off afirst reference beam 25, the means being designed as a first interface21 of a prism 23. At first interface 21, a first reference beam 25 issplit off by partial reflection and strikes position detector 27, whichis designed as a CCD array 29. After passing through first interface 21,first light beam 3 passes through prism 23 and strikes a further meansfor splitting off a second reference beam 33, the further means beingdesigned as a second interface 31. At second interface 31, a secondreference beam 33 is split off by partial reflection and, after totalinternal reflection at a third interface 35 and passage through firstinterface 21, it strikes position detector 27. Located in front of theposition detector is a lens 49, which focuses the reference beams ontoCCD array 29. It is also possible to provide for a slight defocus inorder to achieve a better resolution by interpolation across severalpixels. From the various points of incidence of the reference beams onCCD array 29 it is possible to infer the locations and angles at whichlight beam 3 strikes first interface 21 and second interface 31, andthus the position and propagation direction of light beam 3 afterexiting prism 23. The position detector generates position signals andtransmits them to a processing unit 51. Based on the position datareceived, processing unit 51 drives control elements 13, 17 until thelight beam 5 exiting the prism propagates along the nominal opticalpath, i.e. along nominal axis 89.

The same device can be used to simultaneously align further light beamsto the nominal optical path. Preferably, further control elements areprovided for that purpose.

The present invention has been explained with reference to a specificembodiment. However, changes and modifications can of course be madewithout exceeding the scope of the following claims.

LIST OF REFERENCE NUMERALS

-   1 optical device-   3 first light beam-   5 second light beam-   7 light source-   9 first laser-   11 second laser-   13 first control element-   15 first tilting mirror-   17 second control element-   19 second tilting mirror-   21 first interface-   23 prisms-   25 first reference beam-   27 position detector-   29 CCD array-   31 second interface-   33 second reference beam-   35 third interface-   37 third control element-   39 third tilting mirror-   41 fourth control element-   43 fourth tilting mirror-   45 further first reference beam-   47 further second reference beam-   49 lens-   51 processing work-   53 AOTF-   55 first plane-parallel plate-   57 second plane-parallel plate-   59 optical system-   61 optical fiber-   63 further optical system-   65 illuminating pinhole-   67 beam splitter-   69 scanning mirror-   71 scanning optical system-   73 tube optical system-   75 objective-   77 sample-   79 detection light beam-   81 detection pinhole-   83 detector-   85 PC-   87 monitor-   89 nominal axis

1-20. (canceled)
 21. An optical device for combining a first light beamand at least one second light beam, the optical device comprising: afirst beam splitting device configured to split a first reference beamfrom the first light beam and a second reference beam from the secondlight beam; a second beam splitting device configured to split a thirdreference beam from the first light beam and a fourth reference beamfrom the second light beam; and a position detector configured to detectrespective positions of the reference beams so as to enable at least oneof a respective propagation direction and a respective position of atleast one of the first and second light beams to be adjusted as afunction of at least one of the detected respective positions of thereference beams.
 22. The optical device as recited in claim 21 whereinthe first and second light beams each have a different respectivewavelength.
 23. The optical device as recited in claim 21 wherein thefirst beam splitting device includes a first interface, and the secondbeam splitting device includes a second interface.
 24. The opticaldevice as recited in claim 21 further comprising at least one dispersiveelement.
 25. The optical device as recited in claim 24 wherein thedispersive element includes at least one of a prism, a grating, and anacousto-optical element.
 26. The optical device as recited in claim 21wherein the first and second beam splitting devices are parts of a sameoptical component.
 27. The optical device as recited in claim 26 whereinthe same optical component includes a dispersive element.
 28. Theoptical device as recited in claim 21 wherein at least one of therespective propagation direction and the respective position of thefirst and second light beams are capable of being adjusted independentlyof each other.
 29. The optical device as recited in claim 21 furthercomprising at least one control element configured to adjust at leastone of the respective propagation direction and the respective positionof at least one of the first and second light beams.
 30. The opticaldevice as recited in claim 29 wherein the at least one control elementincludes a tilting mirror.
 31. The optical device as recited in claim 29wherein the at least one control element is configured to be driven as afunction of at least one of the detected respective positions of thereference beams.
 32. The optical device as recited in claim 29 whereinthe at least one control element is disposed upstream of the first beamsplitting device.
 33. The optical device as recited in claim 23 furthercomprising at least one control element configured to adjust arespective angle of incidence of at least one of the first and secondlight beam on the first interface.
 34. The optical device as recited inclaim 23 further comprising at least one control element configured toadjust a respective striking location of at least one of the first andsecond light beams on the first interface.
 35. The optical device asrecited in claim 21 wherein the position detector includes a CCDdetector.
 36. The optical device as recited in claim 21 wherein theposition detector includes a first detector configured to detect therespective position of each of the reference beams.
 37. The opticaldevice as recited in claim 21 wherein the position detector isconfigured to simultaneously detect the reference beams.
 38. The opticaldevice as recited in claim 21 wherein the position detector isconfigured to be calibrated for different respective detectablepositions of the reference beams.
 39. A method for generating anilluminating light beam for a scanning microscope, the methodcomprising: splitting a first reference beam from a first light beam anda second reference beam from a second light beam using a first beamsplitting device; splitting a third reference beam from the first lightbeam and a fourth reference beam from the second light beam using asecond beam splitting device; detecting a respective positions of thereference beams using a position detector; and adjusting at least one ofa respective propagation direction and a respective position of at leastone of the first and second light beams as a function of at least one ofthe detected respective positions of the reference beams.
 40. A scanningmicroscope comprising an optical device for combining a first light beamand at least one second light beam, the optical device comprising: afirst beam splitting device configured to split a first reference beamfrom the first light beam and a second reference beam from the secondlight beam; a second beam splitting device configured to split a thirdreference beam from the first light beam and a fourth reference beamfrom the second light beam; and a position detector configured to detectrespective positions of the reference beams so as to enable at least oneof a respective propagation direction and a respective position of atleast one of the first and second light beams to be adjusted as afunction of at least one of the detected respective positions of thereference beams.